The effects of adult exposure to genistein on female reproductive outcomes

Genistein;Ovary;Follicle;Folliculogenesis;Steroidogenesis;Fertility;Pregnancy;Preconception exposure;Endocrine disruption

Patel, Shreya A

关键词: Genistein;    Ovary;    Follicle;    Folliculogenesis;    Steroidogenesis;    Fertility;    Pregnancy;    Preconception exposure;    Endocrine disruption;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/97673/PATEL-DISSERTATION-2017.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Genistein is a phytoestrogen commonly found in plants such as soybeans, lentils, and chickpeas. Humans and animals are most primarily exposed to genistein through the consumption soy and soy-based dietary products. Human exposure to genistein varies based on diet, but is present in many populations of adults and children. Genistein can bind to and signal through estrogen receptors. Therefore, it has the potential to mimic, enhance, or impair the estradiol biosynthesis pathway, and is categorized as an endocrine disrupting chemical. This is of concern to human health because genistein-induced effects on the steroidogenic pathway can result in adverse, long-term effects on female fertility, cardiovascular, bone, and overall health. The ovary is an estrogen receptor-rich tissue, and plays a key role in the regulation of female fertility. This is because the ovary’s main functional unit, the follicle, is responsible for the production of sex steroid hormones and consists of the oocyte necessary for fertilization. In the adult ovary, the follicle is present in all stages ranging from the immature primordial follicle to the most mature antral follicle. The antral follicle is one of the most important follicle types because it is the primary producer of sex steroid hormones in the ovary, and it is the only follicle type capable of growing to the point of ovulation. Therefore, any effects on folliculogenesis can adversely affect female fertility.Crosstalk between the theca and granulosa cells of the antral follicle is responsible for the production of sex steroid hormones that are necessary for fertility and overall female health. Enzymes in the theca cells metabolize cholesterol to estradiol precursor hormones such as progesterone and testosterone. The androgens can pass the basal membrane and enter the granulosa cells to be further metabolized to estrone and estradiol. Any alteration in this highly regulated enzymatic process could adversely affects steroid hormone production. Normal follicle growth and proper steroid hormone production are necessary to maintain normal female fertility.Thus, I hypothesized that direct exposure to genistein inhibits antral follicle growth and alters the estradiol biosynthesis pathway. To test this hypothesis, I directly exposed intact antral follicles to 6 and 36 µM of genistein for 18-96 hours (h). After each 24 h time point, I measured growth, steroid hormone production, and expression of steroidogenic enzymes. Additionally after 18 h, I measured the expression of apoptotic factors and cell cycle regulators to determine growth inhibition. After 24 h of culture, I found that genistein exposure significantly inhibited antral follicle growth, and that this inhibition continued until the end of the culture period at 96 h. Next, I found that genistein exposure significantly increased the expression of the cell cycle inhibitor Cdkn1a at 18 h. This indicates that direct exposure to genistein inhibits antral follicle growth by causing cell cycle arrest. Furthermore, I found that genistein exposure increased progesterone and DHEA levels, and decreased estrone and estradiol levels. The steroidogenic enzyme expression data indicate that genistein exposure decreased the expression of the enzyme Cyp17a1, the enzyme responsible for further metabolizing progesterone. Thus, explaining the observed increase in progesterone levels and decrease estradiol levels after exposure to genistein. Additionally, I hypothesized that preconception exposure to genistein affects fertility and pregnancy outcomes. To test this hypothesis, female mice were dosed with dietary levels of genistein (300, 500, or 1000 ppm) or a control diet for 30, 60, 150, or 240 days prior to mating. After the dosing period, animals were paired for breeding. This point on, I examined the mice for fertility and pregnancy endpoints, and maternal behavior. I found that after 30 days of dosing, genistein exposure (500 and 1000 ppm) decreased gestation time compared to control. After 60 days of dosing, genistein exposure increased gestation time (500 and 1000 ppm), increased pup mortality (500 and 1000 ppm), decreased litter size (500 ppm), and increased the average pup weight (500 ppm) compared to control. After 150 days of dosing, genistein exposure increased the number of dams with prolonged parturition (300 ppm), pup mortality (300 ppm), and poor maternal behavior (300 ppm). After 240 days of dosing, genistein exposure decreased fertility rates (300 ppm), increased pup mortality (500 ppm), and increased poor maternal behavior (500 ppm). Collectively, these results show that preconception exposure to dietary levels of genistein affects gestation time, increases parturition time, decreases litter size, increases average pup weight, increases pup mortality, and increases poor maternal behavior.Further, I hypothesized that chronic exposure to dietary levels of genistein alters steroid hormone levels and affect ovarian morphology in adult female mice. To test this hypothesis, mice were dosed with genistein (300, 500, or 1000 ppm) or a control diet for 30, 60, 150, and 240 days. At the end of each dosing period, mice were euthanized while in diestrus. At this time, the sera, ovaries, uteri and livers were weighed and collected. The sera were used to measure hormone levels, and the ovaries used for the histological evaluation of follicle numbers. I found that exposure to genistein did not significantly affect progesterone and estradiol levels at any time point. Genistein exposure after 30 days (300 ppm) significantly increased the number of primary follicles compared to control, and exposure to 500 ppm genistein increased the number of primordial follicles compared to control, but this change was of borderline significance. There was no significant change in follicle numbers after 60 days of exposure to genistein. However, mice exposed to genistein for 240 days, then aged to 10 months, had an increase in cystic ovaries compared to control. These results show that genistein exposure does not affect serum hormone levels, has subtle effects on follicle numbers, and increases cystic ovaries in aged mice. Collectively, the results of my doctoral dissertation show that exposure to genistein can adversely affect antral follicle growth and function in vitro, and long term, preconception exposure to environmentally relevant levels of genistein adversely affects fertility and pregnancy outcomes, and ovarian morphology of aged mice.